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Probes for INS

ACD can configure probes for the various manual and automated assays for INS for RNAscope Assay, or for Basescope Assay compatible for your species of interest.

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B cell expansion hinders the stroma-epithelium regenerative cross talk during mucosal healing

Immunity

2022 Dec 13

Frede, A;Czarnewski, P;Monasterio, G;Tripathi, KP;Bejarano, DA;Ramirez Flores, RO;Sorini, C;Larsson, L;Luo, X;Geerlings, L;Novella-Rausell, C;Zagami, C;Kuiper, R;Morales, RA;Castillo, F;Hunt, M;Mariano, LL;Hu, YOO;Engblom, C;Lennon-Duménil, AM;Mittenzwei, R;Westendorf, AM;Hövelmeyer, N;Lundeberg, J;Saez-Rodriguez, J;Schlitzer, A;Das, S;Villablanca, EJ;
PMID: 36462502 | DOI: 10.1016/j.immuni.2022.11.002

Therapeutic promotion of intestinal regeneration holds great promise, but defining the cellular mechanisms that influence tissue regeneration remains an unmet challenge. To gain insight into the process of mucosal healing, we longitudinally examined the immune cell composition during intestinal damage and regeneration. B cells were the dominant cell type in the healing colon, and single-cell RNA sequencing (scRNA-seq) revealed expansion of an IFN-induced B cell subset during experimental mucosal healing that predominantly located in damaged areas and associated with colitis severity. B cell depletion accelerated recovery upon injury, decreased epithelial ulceration, and enhanced gene expression programs associated with tissue remodeling. scRNA-seq from the epithelial and stromal compartments combined with spatial transcriptomics and multiplex immunostaining showed that B cells decreased interactions between stromal and epithelial cells during mucosal healing. Activated B cells disrupted the epithelial-stromal cross talk required for organoid survival. Thus, B cell expansion during injury impairs epithelial-stromal cell interactions required for mucosal healing, with implications for the treatment of IBD.
Engineering an inhibitor-resistant human CSF1R variant for microglia replacement

The Journal of experimental medicine

2023 Mar 06

Chadarevian, JP;Lombroso, SI;Peet, GC;Hasselmann, J;Tu, C;Marzan, DE;Capocchi, J;Purnell, FS;Nemec, KM;Lahian, A;Escobar, A;England, W;Chaluvadi, S;O'Brien, CA;Yaqoob, F;Aisenberg, WH;Porras-Paniagua, M;Bennett, ML;Davtyan, H;Spitale, RC;Blurton-Jones, M;Bennett, FC;
PMID: 36584406 | DOI: 10.1084/jem.20220857

Hematopoietic stem cell transplantation (HSCT) can replace endogenous microglia with circulation-derived macrophages but has high mortality. To mitigate the risks of HSCT and expand the potential for microglia replacement, we engineered an inhibitor-resistant CSF1R that enables robust microglia replacement. A glycine to alanine substitution at position 795 of human CSF1R (G795A) confers resistance to multiple CSF1R inhibitors, including PLX3397 and PLX5622. Biochemical and cell-based assays show no discernable gain or loss of function. G795A- but not wildtype-CSF1R expressing macrophages efficiently engraft the brain of PLX3397-treated mice and persist after cessation of inhibitor treatment. To gauge translational potential, we CRISPR engineered human-induced pluripotent stem cell-derived microglia (iMG) to express G795A. Xenotransplantation studies demonstrate that G795A-iMG exhibit nearly identical gene expression to wildtype iMG, respond to inflammatory stimuli, and progressively expand in the presence of PLX3397, replacing endogenous microglia to fully occupy the brain. In sum, we engineered a human CSF1R variant that enables nontoxic, cell type, and tissue-specific replacement of microglia.
Dynamic human retinal pigment epithelium (RPE) and choroid architecture based on single-cell transcriptomic landscape analysis

Genes & Diseases

2022 Dec 01

Huang, L;Ye, L;Li, R;Zhang, S;Qu, C;Li, S;Li, J;Yang, M;Wu, B;Chen, R;Huang, G;Gong, B;Li, Z;Yang, H;Yu, M;Shi, Y;Wang, C;Chen, W;Yang, Z;
| DOI: 10.1016/j.gendis.2022.11.007

The retinal pigment epithelium (RPE) and choroid are located behind the human retina and have multiple functions in the human visual system. Knowledge of the RPE and choroid cells and their gene expression profiles are fundamental for understanding retinal disease mechanisms and therapeutic strategies. Here, we sequenced the RNA of about 0.3 million single cells from human RPE and choroids across two regions and seven ages, revealing regional and age differences within the human RPE and choroid. Cell-cell interactions highlight the broad connectivity networks between the RPE and different choroid cell types. Moreover, the transcription factors and their target genes change during aging. The coding of somatic variations increases during aging in the human RPE and choroid at the single-cell level. Moreover, we identified ELN as a candidate for improving RPE degeneration and choroidal structure during aging. The mapping of the molecular architecture of the human RPE and choroid improves our understanding of the human vision support system and offers potential insights into the intervention targets for retinal diseases.
Postnatal expansion of mesenteric lymph node stromal cells towards reticular and CD34+ stromal cell subsets

Nature communications

2022 Nov 24

Pezoldt, J;Wiechers, C;Zou, M;Litovchenko, M;Biocanin, M;Beckstette, M;Sitnik, K;Palatella, M;van Mierlo, G;Chen, W;Gardeux, V;Floess, S;Ebel, M;Russeil, J;Arampatzi, P;Vafardanejad, E;Saliba, AE;Deplancke, B;Huehn, J;
PMID: 36433946 | DOI: 10.1038/s41467-022-34868-4

Gut-draining mesenteric lymph nodes (LN) provide the framework to shape intestinal adaptive immune responses. Based on the transcriptional signatures established by our previous work, the composition and immunomodulatory function of LN stromal cells (SC) vary according to location. Here, we describe the single-cell composition and development of the SC compartment within mesenteric LNs derived from postnatal to aged mice. We identify CD34+ SC and fibroblastic reticular stromal cell (FRC) progenitors as putative progenitors, both supplying the typical rapid postnatal mesenteric LN expansion. We further establish the location-specific chromatin accessibility and DNA methylation landscape of non-endothelial SCs and identify a microbiota-independent core epigenomic signature, showing characteristic differences between SCs from mesenteric and skin-draining peripheral LNs. The epigenomic landscape of SCs points to dynamic expression of Irf3 along the differentiation trajectories of FRCs. Accordingly, a mesenchymal stem cell line acquires a Cxcl9+ FRC molecular phenotype upon lentiviral overexpression of Irf3, and the relevance of Irf3 for SC biology is further underscored by the diminished proportion of Ccl19+ and Cxcl9+ FRCs in LNs of Irf3-/- mice. Together, our data constitute a comprehensive transcriptional and epigenomic map of mesenteric LNSC development in early life and dissect location-specific, microbiota-independent properties of non-endothelial SCs.
Identification of visual cortex cell types and species differences using single-cell RNA sequencing

Nature communications

2022 Nov 12

Wei, JR;Hao, ZZ;Xu, C;Huang, M;Tang, L;Xu, N;Liu, R;Shen, Y;Teichmann, SA;Miao, Z;Liu, S;
PMID: 36371428 | DOI: 10.1038/s41467-022-34590-1

The primate neocortex exerts high cognitive ability and strong information processing capacity. Here, we establish a single-cell RNA sequencing dataset of 133,454 macaque visual cortical cells. It covers major cortical cell classes including 25 excitatory neuron types, 37 inhibitory neuron types and all glial cell types. We identified layer-specific markers including HPCAL1 and NXPH4, and also identified two cell types, an NPY-expressing excitatory neuron type that expresses the dopamine receptor D3 gene; and a primate specific activity-dependent OSTN + sensory neuron type. Comparisons of our dataset with humans and mice show that the gene expression profiles differ between species in relation to genes that are implicated in the synaptic plasticity and neuromodulation of excitatory neurons. The comparisons also revealed that glutamatergic neurons may be more diverse across species than GABAergic neurons and non-neuronal cells. These findings pave the way for understanding how the primary cortex fulfills the high-cognitive functions.
Spinal neuropeptide Y Y1 receptor-expressing neurons are a pharmacotherapeutic target for the alleviation of neuropathic pain

Proceedings of the National Academy of Sciences of the United States of America

2022 Nov 16

Nelson, TS;Sinha, GP;Santos, DFS;Jukkola, P;Prasoon, P;Winter, MK;McCarson, KE;Smith, BN;Taylor, BK;
PMID: 36343228 | DOI: 10.1073/pnas.2204515119

Peripheral nerve injury sensitizes a complex network of spinal cord dorsal horn (DH) neurons to produce allodynia and neuropathic pain. The identification of a druggable target within this network has remained elusive, but a promising candidate is the neuropeptide Y (NPY) Y1 receptor-expressing interneuron (Y1-IN) population. We report that spared nerve injury (SNI) enhanced the excitability of Y1-INs and elicited allodynia (mechanical and cold hypersensitivity) and affective pain. Similarly, chemogenetic or optogenetic activation of Y1-INs in uninjured mice elicited behavioral signs of spontaneous, allodynic, and affective pain. SNI-induced allodynia was reduced by chemogenetic inhibition of Y1-INs, or intrathecal administration of a Y1-selective agonist. Conditional deletion of <i>Npy1r</i> in DH neurons, but not peripheral afferent neurons prevented the anti-hyperalgesic effects of the intrathecal Y1 agonist. We conclude that spinal Y1-INs are necessary and sufficient for the behavioral symptoms of neuropathic pain and represent a promising target for future pharmacotherapeutic development of Y1 agonists.
The cancer chemotherapeutic 5-fluorouracil is a potent Fusobacterium nucleatum inhibitor and its activity is modified by intratumoral microbiota

Cell reports

2022 Nov 15

LaCourse, KD;Zepeda-Rivera, M;Kempchinsky, AG;Baryiames, A;Minot, SS;Johnston, CD;Bullman, S;
PMID: 36384132 | DOI: 10.1016/j.celrep.2022.111625

Fusobacterium nucleatum (Fn) is a dominant bacterial species in colorectal cancer (CRC) tissue that is associated with cancer progression and poorer patient prognosis. Following a small-molecule inhibitor screen of 1,846 bioactive compounds against a Fn CRC isolate, we find that 15% of inhibitors are antineoplastic agents including fluoropyrimidines. Validation of these findings reveals that 5-fluorouracil (5-FU), a first-line CRC chemotherapeutic, is a potent inhibitor of Fn CRC isolates. We also identify members of the intratumoral microbiota, including Escherichia coli, that are resistant to 5-FU. Further, CRC E. coli isolates can modify 5-FU and relieve 5-FU toxicity toward otherwise-sensitive Fn and human CRC epithelial cells. Lastly, we demonstrate that ex vivo patient CRC tumor microbiota undergo community disruption after 5-FU exposure and have the potential to deplete 5-FU levels, reducing local drug efficacy. Together, these observations argue for further investigation into the role of the CRC intratumoral microbiota in patient response to chemotherapy.
SERINC5 restricts influenza virus infectivity

PLoS pathogens

2022 Oct 01

Zhao, F;Xu, F;Liu, X;Hu, Y;Wei, L;Fan, Z;Wang, L;Huang, Y;Mei, S;Guo, L;Yang, L;Cen, S;Wang, J;Liang, C;Guo, F;
PMID: 36223419 | DOI: 10.1371/journal.ppat.1010907

SERINC5 is a multi-span transmembrane protein that is incorporated into HIV-1 particles in producing cells and inhibits HIV-1 entry. Multiple retroviruses like HIV-1, equine infectious anemia virus and murine leukemia virus are subject to SERINC5 inhibition, while HIV-1 pseudotyped with envelope glycoproteins of vesicular stomatitis virus and Ebola virus are resistant to SERINC5. The antiviral spectrum and the underlying mechanisms of SERINC5 restriction are not completely understood. Here we show that SERINC5 inhibits influenza A virus infection by targeting virus-cell membrane fusion at an early step of infection. Further results show that different influenza hemagglutinin (HA) subtypes exhibit diverse sensitivities to SERINC5 restriction. Analysis of the amino acid sequences of influenza HA1 strains indicates that HA glycosylation sites correlate with the sensitivity of influenza HA to SERINC5, and the inhibitory effect of SERINC5 was lost when certain HA glycosylation sites were mutated. Our study not only expands the antiviral spectrum of SERINC5, but also reveals the role of viral envelope glycosylation in resisting SERINC5 restriction.
Pitx2 patterns an accelerator-brake mechanical feedback through latent TGFβ to rotate the gut

Science (New York, N.Y.)

2022 Sep 23

Sanketi, BD;Zuela-Sopilniak, N;Bundschuh, E;Gopal, S;Hu, S;Long, J;Lammerding, J;Hopyan, S;Kurpios, NA;
PMID: 36137018 | DOI: 10.1126/science.abl3921

The vertebrate intestine forms by asymmetric gut rotation and elongation, and errors cause lethal obstructions in human infants. Rotation begins with tissue deformation of the dorsal mesentery, which is dependent on left-sided expression of the Paired-like transcription factor Pitx2. The conserved morphogen Nodal induces asymmetric Pitx2 to govern embryonic laterality, but organ-level regulation of Pitx2 during gut asymmetry remains unknown. We found Nodal to be dispensable for Pitx2 expression during mesentery deformation. Intestinal rotation instead required a mechanosensitive latent transforming growth factor-β (TGFβ), tuning a second wave of Pitx2 that induced reciprocal tissue stiffness in the left mesentery as mechanical feedback with the right side. This signaling regulator, an accelerator (right) and brake (left), combines biochemical and biomechanical inputs to break gut morphological symmetry and direct intestinal rotation.
devCellPy is a machine learning-enabled pipeline for automated annotation of complex multilayered single-cell transcriptomic data

Nature communications

2022 Sep 07

Galdos, FX;Xu, S;Goodyer, WR;Duan, L;Huang, YV;Lee, S;Zhu, H;Lee, C;Wei, N;Lee, D;Wu, SM;
PMID: 36071107 | DOI: 10.1038/s41467-022-33045-x

A major informatic challenge in single cell RNA-sequencing analysis is the precise annotation of datasets where cells exhibit complex multilayered identities or transitory states. Here, we present devCellPy a highly accurate and precise machine learning-enabled tool that enables automated prediction of cell types across complex annotation hierarchies. To demonstrate the power of devCellPy, we construct a murine cardiac developmental atlas from published datasets encompassing 104,199 cells from E6.5-E16.5 and train devCellPy to generate a cardiac prediction algorithm. Using this algorithm, we observe a high prediction accuracy (>90%) across multiple layers of annotation and across de novo murine developmental data. Furthermore, we conduct a cross-species prediction of cardiomyocyte subtypes from in vitro-derived human induced pluripotent stem cells and unexpectedly uncover a predominance of left ventricular (LV) identity that we confirmed by an LV-specific TBX5 lineage tracing system. Together, our results show devCellPy to be a useful tool for automated cell prediction across complex cellular hierarchies, species, and experimental systems.
Medial orbitofrontal neurotrophin systems integrate hippocampal input into outcome-specific value representations

Cell reports

2022 Sep 13

Woon, EP;Butkovich, LM;Peluso, AA;Elbasheir, A;Taylor, K;Gourley, SL;
PMID: 36103822 | DOI: 10.1016/j.celrep.2022.111334

In everyday life, we mentally represent possible consequences of our behaviors and integrate specific outcome values into existing knowledge to inform decisions. The medial orbitofrontal cortex (MO) is necessary to adapt behaviors when outcomes are not immediately available-when they and their values need to be envisioned. Nevertheless, neurobiological mechanisms remain unclear. We find that the neuroplasticity-associated neurotrophin receptor tropomyosin receptor kinase B (TrkB) is necessary for mice to integrate outcome-specific value information into choice behavior. This function appears attributable to memory updating (and not retrieval) and the stabilization of dendritic spines on excitatory MO neurons, which led us to investigate inputs to the MO. Ventral hippocampal (vHC)-to-MO projections appear conditionally necessary for value updating, involved in long-term aversion-based value memory updating. Furthermore, vHC-MO-mediated control of choice is TrkB dependent. Altogether, we reveal a vHC-MO connection by which specific value memories are updated, and we position TrkB within this functional circuit.
Clearance of small intestinal crypts involves goblet cell mucus secretion by intracellular granule rupture and enterocyte ion transport

Science signaling

2022 Sep 20

Dolan, B;Ermund, A;Martinez-Abad, B;Johansson, MEV;Hansson, GC;
PMID: 36126118 | DOI: 10.1126/scisignal.abl5848

Goblet cells in the small intestinal crypts contain large numbers of mucin granules that are rapidly discharged to clean bacteria from the crypt. Because acetylcholine released by neuronal and nonneuronal cells controls many aspects of intestinal epithelial function, we used tissue explants and organoids to investigate the response of the small intestinal crypt to cholinergic stimulation. The activation of muscarinic acetylcholine receptors initiated a coordinated and rapid emptying of crypt goblet cells that flushed the crypt contents into the intestinal lumen. Cholinergic stimulation induced an expansion of the granule contents followed by intracellular rupture of the mucin granules. The mucus expanded intracellularly before the rupture of the goblet cell apical membrane and continued to expand after its release into the crypt lumen. The goblet cells recovered from membrane rupture and replenished their stores of mucin granules. Mucus secretion from the goblet cells depended on Ca2+ signaling and the expansion of the mucus in the crypt depended on gap junctions and on ion and water transport by enterocytes adjacent to the goblet cells. This distinctive mode of mucus secretion, which we refer to as "expanding secretion," efficiently cleans the small intestine crypt through coordinated mucus, ion, and fluid secretion by goblet cells and enterocytes.

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Description
sense
Example: Hs-LAG3-sense
Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron#
Example: Mm-Htt-intron2
Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
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Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)
A mixture of multiple probe sets targeting multiple genes or transcripts
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Example: Hs-PDGFB-No-XMm
Does not cross detect with the species (Sp)
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Example: Rn-Pde9a-XMm
designed to cross detect with the species (Sp)
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Example: Mm-Islr-O1
Alternative design targeting different regions of the same transcript or isoforms
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Example: Hs-SLC31A-CDS
Probe targets the protein-coding sequence only
EnEmProbe targets exons n and m
En-EmProbe targets region from exon n to exon m
Retired Nomenclature
tvn
Example: Hs-LEPR-tv1
Designed to target transcript variant n
ORF
Example: Hs-ACVRL1-ORF
Probe targets open reading frame
UTR
Example: Hs-HTT-UTR-C3
Probe targets the untranslated region (non-protein-coding region) only
5UTR
Example: Hs-GNRHR-5UTR
Probe targets the 5' untranslated region only
3UTR
Example: Rn-Npy1r-3UTR
Probe targets the 3' untranslated region only
Pan
Example: Pool
A mixture of multiple probe sets targeting multiple genes or transcripts

Enabling research, drug development (CDx) and diagnostics

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